Folic acid derivatives

ABSTRACT

Novel folic acid derivatives and their use in preparation of γ-esters of folic acid via a pteroyl azide intermediate are described. Folic acid γ-esters are useful intermediates in the synthesis of folic acid conjugates capable of binding folate receptors in vitro and in vivo.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application is a U.S. national counterpart application ofinternational application Ser. No. PCT/US98/21914 filed Oct. 16, 1998,which claims priority to U.S. provisional application Ser. No.60/062,009 filed Oct. 17, 1997.

FIELD OF THE INVENTION

This invention relates to the preparation of folic acid derivatives.More particularly this invention relates to a method of preparingγ-esters of folic acid.

BACKGROUND AND SUMMARY OF THE INVENTION

A recent trend in cancer chemotherapy is the highly aggressiveapplication of high-dose multiple drug treatment regimens at theearliest point of diagnosis. These protocols are limited by drugtoxicity and severe physiological effects and patient fatalities are notuncommon. This situation has caused several members in the medicalcommunity to question whether the benefit/risk boundary has beenexceeded with the agents currently available. Enhancement of thedifferential specificity of anticancer agents by selective targetingmechanisms might diminish such problems. The vitamin folic acid hasattracted considerable attention as a potential means to selectivelydeliver covalently bound drug conjugates. Many human cancer cell lineshave been found to have highly overexpressed membrane-associated folatereceptor proteins which binds folic acid.

Previously, it has been shown that the natural receptor-mediatedendocytosis pathway for the vitamin folic acid can be exploited toselectively and non-destructively deliver folate-conjugated smallmolecules, macromolecules, and drug carriers such as liposomes intocultured tumor cells. When folate is covalently linked to a folatemolecule via its γ-carboxyl moiety, the affinity of the covalentconjugate for the folate cell surface receptor remains essentiallyunaltered. Further, following binding to the cell surface receptor, theconjugated folate is internalized by the cell in much the same manner asthe unmodified vitamin. Recycling of the folate receptor can then leadto further accumulation of the folate conjugates in such target cells.

Unfortunately, a major impediment in design of folate conjugates centersaround synthesis of such compounds. Current practice simply involvestreatment of the substrate of choice with folic acid or a folic acidanalog and a dehydrating agent, such as DCC. This results in a mixtureof both the inactive α-conjugate and the active γ-conjugate oftenaccompanied with the bis-functionalized derivative and/or recoveredfolic acid (see FIG. 1). Separation of these mixtures is often verydifficult.

An alternative approach to the synthesis of differentiallyfunctionalized folic acid derivatives relies upon the acylation ofmonoesters of glutamic acid with pteroic acid. Unfortunately thatstrategy is compromised by the high cost of pteroic acid.

It is an object of the present invention to provide a method forregiospecific substitution of the γ-carboxylic acid functional group offolic acid and related compounds.

There is also provided in the present invention novel pteroic acidderivatives. The glutamic acid moiety of folic acid derivatives iscyclized to form a pyroglutamate group. The pyroglutamate group isnucleophilically displaced to yield pteroic acid derivatives.

This invention also encompasses a method directed to preparing pteroicacid hydrazides by reacting corresponding pyroglutamates with hydrazine.

Another aspect the present invention encompasses a method of preparingγ-substituted folic acid derivatives by reacting the correspondingpteroic azides with γ-substituted glutamate esters.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts a reaction scheme as known in the art for forming folicacid conjugates.

FIG. 2 depicts a reaction scheme for the formation of pyrofolatederivatives from folic acid in trifluoracetic anhydride.

FIG. 3 depicts prior art teaching of reaction of folic acid and eitheracetic or trifluoroacetic anhydride.

FIG. 4 illustrates the reaction of pyrofolate derivatives with ammoniumhydroxide or hydrazine.

FIG. 5 illustrates a comparison of proton nuclear magnetic resonancesignals for structurally related compounds.

FIG. 6 illustrates alternate reaction pathways for the reaction ofpyrofolate derivatives with nucleophiles (Nuc).

FIG. 7 illustrates a reaction scheme for the process of the presentinvention.

FIG. 8 illustrates the preparation of alpha glutamate esters.

FIG. 9 illustrates a reaction scheme for use of γ-glutamate folateesters to prepare γ-substituted folic acid conjugates.

DETAILED DESCRIPTION OF THE INVENTION

In general terms, the present invention is directed toward a method ofpreparing γ-substituted folic acids and related compounds. Theγ-substituted folic acid derivatives are key intermediates for preparingreceptor binding γ-folate-drug conjugates. Current methodology forpreparing folate conjugates result in formation of difficult to separatemixtures of the α-, γ- and bis-functionalized glutamate moiety of folicacid. The present invention provides intermediates and methods for theselective synthesis of folate-drug conjugates linked through theγ-carboxy group on the glutamic acid moiety of folic acid and relatedfolate compounds.

One embodiment of the present invention is a compound of the formula

wherein Q can be a OH or NH₂, Y is either hydrogen, nitroso, C₁-C₄alkyl, C₁-C₄ alkanoyl, or a C₁-C₄ halosubstituted alkanoyl, includingdi- and tri-halosubstituted alkyl, and Z is either hydrazino or apyroglutamate group, provided that when Z is pyroglutamate, Y is otherthan acetyl or trifluroacetyl.

The term C₁-C₄ alkyl includes a straight, branched or cyclic aliphaticchain having one to four carbon atoms. Typical C₁-C₄ alkyl groupsinclude methyl, ethyl, propyl, isopropyl, butyl, iso-butyl, andtert-butyl.

The term C₁-C₄ alkanoyl includes ethanoyl, propanoyl, isopropanoyl,butanoyl, and t-butanoyl.

This invention is also directed to a method of preparing a compound ofthe formula

wherein Q and Y are defined as above. The method comprises the step ofreacting the corresponding pyrofolate (Formula I above, Z=pyroglutamate)with hydrazine, provided that when Y in the pyrofolate istrifluroacetyl, Y in the product hydrazide of Formula II is hydrogen.

In another embodiment, this invention is also directed to a method ofpreparing a γ-substituted compound of the formula

wherein Q is —OH or NH₂, and Y is hydrogen, or C₁-C₄ alkyl and R isalkyl. The method comprises the step of reacting a compound of theformula

with an γ-alkyl glutamate in DMSO or DMF in the presence of a base.

Thus there is provided in accordance with this invention a method forpreparation of γ-substituted folate derivatives. The initial step ofthis methodology involves the formation of a pyrofolate. TheN10-trifluoroacetyl derivative of azlactone 8′ (not the isomericpyrofolate 8; see FIG. 2) has been alleged to result from reaction offolic acid 1 with trifluoroacetic anhydride (See FIG. 3). In addition,it has been reported that N10-acetyl aziactone Me-8′ results from theanalogous acetic anhydride reaction. (Temple, Jr., C.; Rose, J. D.;Montgomery, J. A. J. Org. Chem., 46, 3666 (1981). Basic hydrolysis ofMe-8′ yields a mixture of folic acid 1 and pteroic acid 10, that isentirely consistent with the pyrofolate structure while an theunprecedented hydrolysis of the azlactone imine moiety had to be invokedto explain the production of pteroic acid. This structural misassignmenthas dire consequences vis-a-vis regiospecific functionalization of thetwo carboxylates of the glutamate moiety, since nucleophilic attack onthe azlactone 8′ would be expected to afford functionalization of theα-carboxyl moiety while activation of the carboxylic acid should enableregiospecific γ-functionalization. This is exactly the oppositeregiochemistry that actually results from functionalization of thecorrect pyrofolate structure 8, and has resulted in further incorrectstructural assignments.

Regiospecific functionalization of the γ-carboxylate of glutamic acid isroutinely accomplished using urethane derivatives of pyroglutamic acidwhich exploit the γ-lactam moiety as an acylating agent. A search of theliterature surprisingly revealed that pyrofolic acid 9 or its N-acylatedderivatives are apparently unknown. Nevertheless, treatment of folicacid 1 with excess trifluoroacetic anhydride in THF for 10 h from 0° C.to 25° C. produces N2,10-bistrifluoroacetyl pyrofolic acid 7 as anextremely water-labile material which is believed to be a mixture ofdiacylated anhydride 6 and diacylated carboxylic acid 7 as judged by¹⁹F-NMR of the crude reaction mixture. In any event, simply stirring theaforementioned material with neutral water affords N10-trifluoroacetylpyrofolic acid 8 in a quantitative yield. (See FIG. 2). Compound 8 canbe further transformed to pyrofolic acid 9 via deacylation with cesiumcarbonate and water (87%). Both compounds 8 and 9 are essentiallyracemic as determined using an enzymatic assay based uponcarboxypeptidase G.

In order to provide a definitive structural assignment, bothN10-trifluoroacetyl pyrofolic acid 8 and pyrofolic acid 9 were treatedwith excess concentrated ammonium hydroxide at 25° C. for several hoursto afford pteroyl amide 11 along with pyroglutamic acid 12 in high yield(See FIG. 4), a finding which would be exceptionally difficult torationalize with the alternative azlactone structures 8′ and 9′. Asexpected, concomitant deacylation of the N10-trifluoroacetyl moietyoccurred during the reaction. The isolated pyroglutamic acid 12exhibited only ≦5% optical activity in accord with extensiveracemization occurring during the synthesis of 8. Monitoring a pair ofsimilar reactions of 8 and 9 with hydrazine (10 equiv.) in DMSO-d₆ at25° C. for 9 h unambiguously produces a 1:1 mixture of pteroyl hydrazide13 and pyroglutamic acid 12 in near-quantitative yield as assayed by¹H-NMR, and HPLC (FIG. 4 and Table 1, Entries 12, 13).

TABLE 1 Reactions of Nucleophiles with N¹⁰-Trifluoroacetylpyrofolic acid8 and pyrofolic acid 9 Folic Acid Pteroic Acid Nucleophile andDerivative 25 Derivative 26 Folate/Pteroate Run SM Conditions (yield^(a)& Nuc=) (yield^(a) & Nuc=) (25/26) Ratio^(a) 1 8 H₂O, NaOH 25a = 1 (52%,OH) 26a = 10 (48%, OH) 1.1 2 9 H₂O, NaOH 25a = 1 (68%, OH) 26a - 10 (32,OH) 2.1 3 8 H₂O, HCl 25b^(b) (33%, OH) 26b^(b) (56%, OH) 0.5 4 9 MeOH,NaOMe 25c (49%, OMe) 26c (44%, OMe) 1.1 5 9 MeOH, LiOMe 25c (81%, OMe)26c (19%, OMe) 4.3 6 9 MeOH, DBU 25c (58%, OMe) 26c (42%, OMe) 1.4 7 8MeOH, TlOMe 25c (35%, OMe) 26c (58%, OMe) 0.6 8 9 MeOH, TlOMe 25c (69%,OMe) 26c (30%, OMe) 2.3 9 9 i-PrOH, TlOi-Pr 25d (80%, O-iPr) 26d (7%,O-iPr) 11.5 10 9 t-BuOH, TlOt-Bu 25e (Nrc, Ot-Bu) 26e (Nr^(c), Ot-Bu)NR^(c) 11 9 DEG^(d), NaH 25f (48%, DEG^(e)) 26f (22%^(a), 2.2 DEG^(e))12 8 NH₂NH₂, DMSO 25g (0.9%, 26g = 13 (91%, 0.01 NHNH₂) NHNH₂) 13 9NH₂NH₂, DMSO 25g (9.1%, 26g = 13 (90%, 0.1 NHNH₂) NHNH₂) 14 8 NH₄OH, H₂O25h (33%, NH₂) 26h = 11 (67%, 0.5 NH₂) 15 9 NH₄OH, H₂O 25h (60%, NH₂)26h - 11 (40%, 1.5 NH₂) 16 8 PMBA^(f) 25I (0.9%, 26i (75%, 0.01PMBA^(g)) PMBA^(g)) ^(a)Product ratio assayed by HPLC; ^(b)This is theonly instance in which the N¹⁰-trifluoroacetyl moiety survived thereaction conditions; ^(c)NR = No reaction; ^(d)DEG = HOCH₂CH₂OCH₂CH₂OH;^(e)DEG′ = OCH₂CH₂OCH₂CH₂OH; ^(f)PMBA - p-methoxybenzylamine;^(g)PMBA′ - p-methoxybenzylamino.

Spectral evidence strongly supports the pyrofolate structure assigned tocompounds 8 and 9. In particular, the ¹³C NMR shift (DMSO-d₆) of themethine-bearing chiral carbon is highly diagnostic (FIG. 5). Folic acid1 and the truncated p-aminobenzoyl glutamates 14-16, all resonate at˜52γ. Pyrofolates 8 and 9 and model p-aminobenzoyl pyroglutamates 17-21,exhibit their methine carbons between 59-60γ. This can be contrasted tomodel azlactones 22-24, which have chemical shifts around 64-66γ.

Having secured the pyrofolate structure of derivatives 8 and 9, theregiospecificity of the reaction of these substrates with oxygen andnitrogen nucleophiles was investigated. While the pyrofolate moietyinsures complete regioselection between the α and γ-carbonyl groups offolic acid, the problem of differentiating between the two imidecarbonyl groups (γ-CO and Pte-CO) of 8 and 9 remained to be determined.As can be seen in Table 1, Thallium[I]-mediated alcoholysis providespreferential generation of the folic acid derivatives 25c,d, theselectivity is an important function of the stearic environment of thealcohol. Aminolysis is currently unacceptable for direct drugconjugation, but as will be seen in FIG. 7, acyl hydrazide 26g, compound13, is an ideal intermediate for the indirect synthesis of folates vianitrogen acylation of glutamates with pteroylazide 27. As expected,N10-trifluoroacetyl derivative 8 consistently exhibits a smallerfolate/pteroate ratio (25/26) relative to compound 9 since increasednucleophilic attack at the benzoate carbonyl moiety is favored when thep-amino lone pair is unavailable for resonance deactivation. The fullmagnitude of this effect cannot be accurately assessed simply byinspection of Table 1, since there was no attempt to determine the rateof deacylation of the N10-trifluoroacetyl group relative to nucleophilicattack at the two competing carbonyl groups (See FIG. 6).

Since it had already determined that pyrofolic acid derivatives 8 and 9were essentially racemic, an indirect synthesis of the desiredγ-functionalized folic acid derivative 3 shown in FIG. 1 was pursued viaexploitation of the exceptionally selective (>99:1) reaction ofhydrazine with compound 8 (Table 1, Entry 12). To this end, it wasobserved that conversion of pteroyl hydrazide 13 to pteroylazide 27 canbe conveniently effected on reasonable (28 g) scale simply by treatmentwith 1.0 equivalent of t-butyl nitrite in trifluoroacetic acidcontaining 5 mol % potassium thiocyanate for 4 h at 10° C. HPLC analysisreveals that reactions run in the absence of potassium thiocyanate, anN-nitroso transfer catalyst, also produce 20-30% of N10-nitrosopteroylazide 28 along with pteroylazide 27. In the KSCN-free conditionthe remaining pteroyl hydrazide 13 slowly reacts with 28 to affordadditional amounts of 27, but the reaction produces unacceptable levelsof impurities relative to the optimized procedure.

Assay of pteroylazide 27 by UPLC reveals a purity of 91%. The knownimpurities include ˜4% pteroic acid 10, ˜2% of the aniline (PteNH₂)apparently resulting from Curtius rearrangement of 27, ˜2% ofpteroylamide (PteCO—NH₂ 11); and <1% of folic acid 1. Pteroylazide 27may be further purified by dissolution/precipitation fromtrifluoroacetic acid/isopropanol, but the known impurities do notinterfere, and the crude ˜90% pure material can be used routinely forvirtually all coupling operations. Unlike some acyl azides, pteroylazide27 is quite stable; samples of the lemon-yellow solid may be stored inthe dark in a freezer for at least 12 months without appreciabledecomposition. Room temperature samples of 27 protected from lightappear to have shelf lives of at least a month; while samples of 27darken appreciably when exposed to light, their HPLC profiles are notsubstantially degraded.

Therefore, pteroylazide 27 is now both easily available and serves as anexcellent reagent for nitrogen acylation of differentially protectedglutamates. As can be seen in Table 2, reaction of pteroylazide 27 inDMSO with L-glutamic acid 30d or glutamates 30a-c is strongly influencedby the nature of the added base, tetramethylguanidine 39a (TMG),t-butyltetramethylguanidine 39b (BTMG), and the expensiveN-Methyl-1,5,9-Triazabicyclo[4.4.0]decene 38 (MTBD) all giving superbreactions. Our initial hypothesis is that the more basic nature of theguanidine bases, in concert with their ability to form solubleguanidinium carboxylates, is responsible for their ability to foster theacylation reaction. Surprisingly, it is noted that both Rosowsky andChaykovsky (Rosowsky, A., et al., J. Med. Chem., 24, 1450 (1981);Rosowsky, A., et al., J. Med. Chem., 25, 960 (1982); Chaykovsky, M., etal., J. Med. Chem., 18, 909 (1975); Chaykovsky, M., et al., J. Med.Chem., 22, 869 (1979); Chaykovsky, M., et al., J. Med. Chem., 22, 874(1979)) have reported that the closely related acyl azide 29 isinsufficiently reactive (in DMF or DMAC) to acylate glutamates or otherα-aminoesters.

TABLE 2 Acylation of glutamic Acid Derivatives 30-a-d in DMSO with AcylAzides 27, 29, 31, 32 Acyl Azide Base (# eq); HPLC & DMSO Product,conversion, Run Glutamate pKa Conditions and/or (isolated yield) 1 27 +L-30a Et₃N (3 eq); 48h, 40° C. L-33a  23% 18.5 2 27 + L-30a Et₃N (5 eq);48h, 40° C. L-33a  25% 18.5 3 27 + L-30a TMG  6h, 25° C. L-33a 100%(88%) (2 eq); 20 4 27 + L-30a MTBD 12h, 25° C. L-33a 100% (2 eq); 20 527 + L-30a BTMG  9h, 25° C. L-33a 100% (2 eq); 21 6 27 + L-30a DBU  8h,25° C. L-33a  77% (2 eq); 19 7 27 + L-30b TMG 10h, 25 ° C. L-33b 100%(60%) (2 eq); 20 8 27 + L-30c TMG 12h, 25° C. L-33c 100% (82%) (2 eq);20 9 27 + L-30d TMG  9h, 25° C. L-25a = L-1 100% (67%) (3 eq); 20 1027 + L-30d TMG  4h, 25° C. D-25a = D-1 100% (40%) (3 eq); 20 11 27 + D,TMG  3h, 25° C. D, L-25a = D, 100% (38%) L-30d (3 eq); 20 L-1 12 29 +L-30d TMG  1h, 25° C. L-34d (100%) (3 eq); 20 13 31 + L-30d TMG  1h, 25°C. L-35d  (73%) (3 eq); 20 14 31 + L-30a TMG  1h, 25° C. L-36a - 15 (67%) (2 eq); 20 15 32 + L-30a TMG  1h, 25° C. L-37a = 16  (72%) (2eq); 20

Examples of other bases suitable for use in accordance with thisembodiment of the present invention include trimethylamine,triethylamine, tripropylamine, diisopropylamine, diisopropylethylamine,N-methylmorphine, tetramethyl piperidine,1,5-diazabicyclo[4.3.0]non-5-ene (DBN),1-8-diazabicyclo[5.4.0]undec-7-ene (DBU); dicyclohexylamine (DCHA),[1.8-bis(dimethylamino)naphthalene]dimethylethylamine; tetramethylguanidine (TMG) t-butyl tetramethyl guanidine (BTMG), N-methyl,1,5,9-triazobicyclo[4.4.0]undecene (MTBD), and 2,6-lutidine.

The dramatic reactivity difference between 27 and 29 appears to be aconsequence of abstraction of the N—H proton of the p-aminobenzoyl azidemoiety by the guanidine base followed by 1,6-elimination of the azideanion to generate the neutral p-quinoketene monoamine analog of 27′ (SeeFIG. 7: Y=lone pair; no charge on N). Presumably such a species would bean exceptionally reactive acylating agent. Two facts mitigate againstthis intriguing possibility: (1) The pKa of the N—H proton of 27 shouldbe similar to p-methylamino methylbenzoate, having a pKa of 25.7 inDMSO, which is fairly distant from the pKa of H-TMG+ at 13.6 in water(est. ˜20 in DMSO); consequently the concentration of the deprotonatedform of 27 would be relatively small, but still attainable; (2) A secondobservation which argues against 27′ (FIG. 6: Y=lone pair, no charge onN) being a requisite intermediate is that azide 29 which bears no N—Hproton, is a perfectly fine acylating agent, providing a high yield ofsynthetic L-methotrexate 34d upon reaction with L-glutamic acid 30d andtetramethylguanidine 39a, provided that DMSO or DMF is employed as thereaction solvent (Table 2, Entry 12). The fact that 29 reacts morerapidly with 30d than does 27 rules out the possibility that N—Hdeprotonation is an integral feature in these acylation reactions.Furthermore, there is no special reactivity conferred to these acylazides by the pterion moiety, since p-monomethylaminobenzoyl azide 31and p-dimethylaminobenzoylazide 32 both react with γ-methylglutamate 30ain DMSO or DMF in the presence of tetramethylguanidine to afford N-acylglutamates 36a and 37a, respectively. The question as to whether theseacylations are simply proceeding via the standard tetrahedral adduct ormay progress via the intermediacy of p-quinoketene monoiminium ions 27′,29′, 31′ and 32′ still remains to be determined (See FIG. 7).

Control studies show that in the absence of an added substrate,tetramethylguanidine (TMG) 39a slowly reacts with pteroylazide 27 orp-methylaminobenzoyl azide 31 to afford acylated guanidines 40 or 41(See FIG. 7), but HPLC analyses of the reactions of 27 with glutamates30a-d show only traces of compound 40. By comparison, HPLC analysis of aDMSO solution of 27 with pentaalkylguanidines BTMG 39b and MTBD 38 givesno evidence of guanidine acylation or any other reaction after 18 h at25° C. This finding requires that, if formed, p-quinoketene monoiminiumion 27′ must be in ready equilibrium with the starting acyl azide 27.DBU (est. pKa in DMSO ˜19) 20c was found to be a less effective base inthese reactions (Table 2, Entry 5), since a portion of the pteroylazide27 was consumed via an unprofitable acylation-fragmentation sequenceproviding by-product 45 in 20% yield (see FIG. 8).

Since it had been previously observed that guanidine bases are capableof racemizing acylated glutamates (under more forcing conditions thanwere employed in the reactions listed in Table 2), the optical purity ofthe folic acid 1 (=25a) and several of its derivatives which had beenproduced was evaluated. For this purpose it was elected to employ theenzyme carboxypeptidase G, which has been shown to only deacylate thenatural L-enantiomer of folic acid 1 to L-glutamic and pteroic acid 10,the yield of the latter material being determined by HPLC. In order toassay the pyrofolates 8 and 9 as well as the α- and γ-monoesters offolic acid L-33a and L-33c, an initial mild basic hydrolysis wasrequired prior to the enzyme assay. As can be seen in Table 3, Entries1,2, the synthetic folic acid L-1=25a was produced in enantiomericallypure form (within the limits of experimental error). Control reactionson synthetic D-1=25a and DL-1=25a (Table 3, Entries 3,4) serve toconfirm the error limits of the enzymatic method at about ±2-3%.

Synthesis of bis alkylated glutamates 51a,b was initiated via reactionof L-glutamic acid 30d with allyl chloroformate to affordN-alloc-protected glutamic acid 47. This material was not purified butdirectly reacted with paraformaldehyde under the standard conditions 16to provide N-alloc oxazolidinone 48 in 50% overall yield for the twosteps. Reaction of this substrate with slightly less than 2 equivalentssodium metal in the presence of an excess of allyl 49 or dimethylallylalcohol 50 provides α-alkylated and α-dimethylallylated N-allocglutamates 51a,b, in 79% and 71% yield, respectively. Use of an excessof sodium results in partial racemization of glutamate 51b; in thisinstance 2.1 equivalents of sodium generated a product which was only85.9% optically pure (Table 3, Entry 10a), while employment of the 1.9equivalent conditions afforded material which was 96.7% optically pureas judged by the enzymatic assay (Table 3, Entry 10b). Since thesynergistic value of Palladium [0]-mediated deprotection of the allyland dimethylallyl protecting groups has been convincingly demonstratedfor N-alloc esters. This superb strategy was used for cleavage of 51a,b.As can be seen in FIG. 8, reaction of 51a with diethylamine and 10 mol %Palladium tetrakistriphenylphosphine provides a quantitative yield ofL-glutamic acid 30d, while similar reaction of dimethyl allyl ester 51baffords a 70% yield of α-DMA protected glutamate 30c. As indicatedabove, both these materials are shown to be essentially optically pure,as assayed after their conjugation (69-82%) with pteroyl azide 27, toafford 25a (=L-1) and L-33c, respectively (Table 3, Entries 3, & 10b).

The value of pteroylazide 27 for the synthesis of folic acid analogs isexemplified in the preparation of DTPA-folate(γ) 53, a metal-bindingligand of an important new imaging agent with outstanding tumorspecificity (see FIG. 9). The initial sample of 52, the precursor of 53,was prepared via the direct DCC/NHS coupling of ethylenediamine withfolic acid 1 followed by separation of the various reaction componentsby extensive HPLC, the final yield of the requisite γ-conjugate 52 (FIG.1 compound 3; X−Z=NHCH₂CH₂NH₂) being on the order of 10-15% on smallscale. The current synthesis of 52, which is far superior in practicalterms, now simply involves the reaction of synthetic γ-methylfolateL-33a (Table 2, Entry 3, 1.1 equiv) with neat ethylenediamine (50 equiv)for 3 h at 25° C. to afford 52 as a yellow solid in 87% yield, withoutthe need of chromatography. Conversion of 52 to the tumor-specificmetal-binding ligand DTPA-folate(γ) 53 has been previously described.

In conclusion, the first efficient syntheses of racemicN10-trifluoroacetyl pyrofolic acid 8, racemic pyrofolic acid 9,pteroylhydrazide 13, and pteroylazide 27 is provided. The latter reagentis an effective and economic reagent for the synthesis ofdifferentially-functionalized folic acid derivatives. Application ofthis technology for the synthesis of new folate anticancer drugconjugates is under active investigation.

TABLE 3 Carboxypeptidase G anantiospecific hydrolysis of folic acidderivatives. Run Compound (Source) % Hydrolysis 1 L-1 (commercial^(a))96.7 2 L-25a = L-1 (from Table 2) 98.4 3 L-25a = L-1 (from Table 3) 98.64 D-25a = D-1 (from Table 2)  3.0 5 D, L-25a = D, L-1 (from Table 2)52.3 6 L-34d (NCI MTX^(b)) 96.1^(c) 7 L-34d (MTX from Table 2) 99.2^(c)8 DL-33a (from Table 1) 54.4 9 L-33a (from Table 2) 96.8 10a L-33c (fromTable 2) 85.9 10b L-33c (from Table 2 & Scheme 7) 96.7 11 8 (from Scheme2) 49.5 12 9 (from Scheme 2) 51.7 ^(a)Commercial L-1 from Vitamins,Inc.; ^(b)A sample of L-methotrexate L-34d was provided by the NationalCancer Institute; ^(c)D-34d is known not to be hydrolyzed bycarboxypeptidase G.

EXPERIMENTAL SECTION

General Methods. Melting points were obtained on a MEL-TEMP apparatusand are uncorrected. Unless otherwise stated, reactions were carried outunder argon in flame dried glassware. Tetrahydrofuran (THF) and diethylether (Et₂O) were distilled from sodium-benzophenone ketyl.Dichloromethane and benzene were distilled from calcium hydride.Cyclohexane was stored over sodium metal. Deuterated NMR solvents (CDCl₃and CD₃CN) were stored over 4 Å molecular sieves for several days priorto use. Flash chromatography on silica gel was carried out (230-400 meshsilica gel was used), and reverse phase LC was used for preparativepurpose (LiChroprep C-18, 310 mm×25 mm). ¹H and ¹³C NMR spectra wereobtained using GE QE-300 NMR and Varian Gemini 200 NMR spectrometers at300 or 200 MHz and 75 or 50 MHz respectively. ¹H NMR chemical shifts arereported in ppm relative to the residual protonated solvent resonance:CDCl₃, γ 7.26; C₆D₅H, γ 7.15. Splitting patterns are designated as s,singlet; d, doublet, t, triplet; q, quartet; p, pentet; m, multiplet;br, broadened. Coupling constants (γ) are reported in Hz. ¹³C NMRchemical shifts are reported in ppm relative to solvent resonance:, γ77.00; C₆D₆, γ 128.00. Mass spectral data were obtained on a Finnigan4000 mass spectrometer (low resolution) and a CEC 21 110 B highresolution mass spectrometer, with the molecular ion designated as M.All the chemicals were supplied by Aldrich Chemical Company, Inc,Milwaukee, Wis. 53233, or otherwise indicated.

EXAMPLE 1

Typical experimental procedure for coupling of pteroylazide 27 withglutamates:

To a stirred DMSO suspension of equal molar amounts of pteroylazide 27(0.02˜0.3M) and a glutamate (Table 2) was added two (three for glutamicacid) equivalents of tetramethylguanidine at room temperature. Themixture soon became homogeneous, and the reaction was normally completewithin 12 h as indicated by analytical HPLC. The solution was filteredthrough a pad of celite to remove any traces of solid residue, andacetonitrile was slowly added to the stirred filtrate. The precipitatedsolid was filtered centrifuged to give the crude product after washingwith diethyl ether and drying 18 h under vacuum. When appropriate,preparative LC was used to provide a purer sample. The procedure isexemplified below by the synthesis of folic acid L-1 andtetramethylguanidinium (L)-methyl folate (γ) 33a.

EXAMPLE 2

Synthetic Folic acid L-1 (=25a):

To a suspension of pteroylazide 27 (200 mg, 0.590 mmol) and L-glutamicacid (131 mg, 0.890 mmol) in DMSO (30 mL) was added neattetramethylguanidine (0.222 mL, 1.77 mmol). The stirred mixture soonbecame homogeneous, and the reaction was complete after 9 h as indicatedby analytical HPLC. The solution was filtered through a pad of celite toremove any traces of solid residue, and acetonitrile (50 ml) was slowlyadded to the stirred filtrate. The precipitated solid was thencentrifuged to give the crude folic acid (248 mg) after washing withaqueous 1% HCl solution (10 mL×1), acetonitrile (10 mL×1), and diethylether (25 mL×2), and drying 18 h under vacuum. An analytical sample (174mg, 67%) was obtained using preparative reverse phase HPLC. ¹H NMR, UV,and analytical HPLC, all indicated the same as those of commercial folicacid. Decomp. point ˜238° C. [α]25D=+17.4° (c=0.5 in 0.1N NaOH); 98.4%of L-folic acid by the enzyme assay. Analytical HPLC Rt=7.2 min (Flowrate: 0.7 mL/min; Eluent A: water and phosphate buffer 50 mmol/dm3, pH7; Eluent B: acetonitrile; Gradient: 0 min, 2% B; 25 min, 50% B; Column:ECONOSPHERE C18, 150 mm×4.6 mm).

EXAMPLE 3

Determination of optical purity of folate derivatives listed in Table 3using enzymatic hydrolysis with carboxypeptidase G:30:

The folic acid derivative (˜2.5 mg) was dissolved in 1 mL Tris buffercontaining 20 μg ZnCl2 and 50 microunit enzyme (Sigma Chemical Company)was added. The solution was incubated at 37° C. for 2 h. A 20 μL samplewas tested by HPLC on a 4.6 m×250 mm MICROSORB C-18 reverse phase column(Eluent A: 5 mM phosphate buffer; Eluent B: acetonitrile; flow rate: 1mL/min; gradient: 0-5 min, 5% B, 10-15, 25% B). Folic acid was eluted at4.1 min, pteroic acid at 10.6 min, methotrexate at 10.8 min, andamino-N10-methyl pteroic acid at 11.9 min. Peak area was used as thestandard for analysis.

For folate derivatives, ˜5 mg was first dissolved in 1 mL 0.25 M NaOHand incubated at room temperature for 1 h. Then the solution wasacidified by 1 M HCl until precipitation. After centrifugation, washing,and drying, the ˜2.5 mg yellow pellet was dissolved in 1 mL Tris bufferand reacted with enzyme under the conditions described above.

EXAMPLE 4

N2,10-bis-Trifluoroacetylpyrofolic acid/anhydride 6/7:

To a mechanically stirred suspension of folic acid 1 (100 g, 0.23 mol,U.S.P. grade, supplied from Vitamins Inc., Chicago, Ill., 60601) andanhydrous tetrahydrofuran (1,000 mL) in a three-neck flask,trifluoroacetic anhydride (256 mL, 1.81 mol) was slowly added at 0° C.over ˜0.5 h before warming the solution to 25° C. The mixture graduallyturned into a dark brown homogeneous phase as the reaction proceeded.After 10 h, analytical HPLC showed that the reaction was complete by theappearance of a number of peaks (presumably a mixture of 7 and othertrifluoroacetylated pyrofolic acids) and confirmed the absence of folicacid 1. The solution was filtered through a pad of celite to remove asmall amount of solid residue. Using a rotary evaporator, the filtratewas concentrated to a dark brown viscous liquid (˜300 mL), which wasslowly transferred with an aid of tetrahydrofuran (˜20 mL) to a flask ofwell-stirred benzene (1,500 mL). The precipitated yellowish solid wasfiltered and washed with diethyl ether (250 mL×1) to yield crude product6/7 (151 g). ¹H NMR (300 MHz, DMSO-d₆) γ 8.89 (s, 1H, C7-H), 7.65 (s,4H, Ar), 5.25 (s, 2H, C9-H2), 4.71 (dd, J=4.2, 8.9 Hz, C19-H), 2.59˜1.98(overlap, 4H). ¹³C NMR (200 MHz, DMSO-d₆) γ 174.4, 172.6, 170.9, 169.1,166.0, 165.3, 159.8, 159.1, 159.0, 158.3, 157.5, 156.4, 155.7, 155.1,149.2, 147.4, 142.2, 135.1, 130.1, 129.4, 128.4, 124.9, 124.8, 119.2,118.1, 113.5, 112.4, 107.7, 106.6, 58.7, 54.1, 31.4, 21.6. ¹⁹F NMR (300MHz, DMSO-d₆) γ −65.66, −74.13, −80.47 (Integration ratio1.0:0.93:0.13). Analytical HPLC Rt=12.7 min (Flow rate: 0.7 mL/min;Eluent A: water and phosphate buffer 50 mmol/dm3, pH 7; Eluent B:acetonitrile; Gradient: 0 min, 2% B; 25 min, 50% B; Column: ECONOSPHEREC18, 150 m×4.6 mm).

EXAMPLE 5

N10-Trifluoroacetylpyrofolic acid 8:

The crude N2,10-bis-trifluoroacetylpyrofolic acid/anhydride 6/7 (150 g)was dissolved in tetrahydrofiran (500 mL), followed by addition of ice(˜100 g) with stirring. Analytical HPLC indicated that all the originalpeaks converged after ˜3 h at 25° C. into a single one(N10-trifluoroacetylpyrofolic acid 8). The mixture was then slowlytransferred to efficiently stirred diethyl ether (2,000 mL). Theprecipitated yellowish powder was filtered, triturated with diethylether, washed thoroughly with diethyl ether (200 mL×3), and dried 18 hunder vacuum, giving 8 (123 g) in a quantitative yield from folicacid 1. ¹H NMR (300 MHz, DMSO-d₆) γ 8.64 (s, H, C7-H), 7.62 (s, 4H, Ar),5.12 (s, 2H, C9-H2), 4.70 (dd, J=3.2, 4.9 Hz, 1H, C 19-H), 2.54˜2.43(overlap, 4H). ¹³C NMR (300 MHz, DMSO-d₆) γ 176.8, 174.7, 172.9, 169.4,161.2, 156.9, 156.4, 156.0, 155.8, 155.5, 154.5, 149.8, 149.7, 145.1,142.5, 135.3, 130.3, 128.7, 118.5, 114.7, 110.9, 59.0, 54.4, 31.8, 21.9.¹⁹F NMR (300 MHz, DMSO-d₆) γ −65.2. mass spectrum (FAB), 519 (MH+); HRMScalcd for C₂₁H₁₆F₃N₇O₆ 519.1114, found 519.1112. Softens and decomp. at208˜214° C. Elemental analysis calcd for C₂₁H₂₆F₃N₇O₆·.0.5H₂O H 3.24, C47.73, F 10.79, N 18.65; found H 3.04, C 47.63, F 10.72, N 18.64.[α]D25=+2.9,c=0.5 in DMSO (cf. commercial folic acid from Vitamins, Inc.=+14.4 at the same concentration). Analytical HPLC Rt=11.9 min (Flowrate: 0.7 mL/min; Eluent A: water and phosphate buffer 50 mmol/dm3, pH7; Eluent B: acetonitrile; Gradient: 0 min, 2% B; 25 min, 50% B; Column:ECONOSPHERE C18, 150 m×4.6 mm).

EXAMPLE 6

Pyrofolic acid 9:

To a stirred homogeneous solution of N10-trifluoroacetylpyrofolic acid 8(14 g, 27 mmol) and DMF (250 mL) was slowly added aqueous cesiumcarbonate (10 mL, 82 mmol) at 25° C. Analytical HPLC indicatedcompletion of the reaction by disappearance of 8 in 5 h. The mixture wasfiltered through a pad of celite, and carefiilly acidified to pH 4 with5% aqueous hydrochloric acid. The resulting precipitate was thoroughlywashed with water (100 mL×3) by centrifigation, acetonitrile (100 mL×1),and diethyl ether (100 mL×2) by aspirator filtration. The yellowishproduct 9 (10.2 g, 89%) was obtained after drying 24 h at 60° C. undervacuum. ¹H NMR (300 MHz, DMSO-d₆) γ 8.64 (s, H, C7-H), 7.41 (d, J=8.3Hz, 2H, Ar), 6.58 (d, J=8.3 Hz, 2H, Ar), 4.70 (m, 1H, C19-H), 4.47 (d,J=4.7 Hz, 2H, C9-H2), 2.57˜1.80 (overlap, 4H). ¹³C NMR (300 MHz,DMSO-d₆) γ 174.0, 172.8, 169.1, 161.2, 153.9, 152.2, 148.7, 148.4,132.4, 132.3, 128.1, 120.4, 110.6, 59.0, 45.8, 31.6, 21.6. mass spectrum(FAB), 424 (MH+); HRMS calcd for C₁₉H₁₇N₇O₅ 424.1369, found 424.1365.Mp: decomp. at ˜269° C. [α]D25=−1.2,c=0.5 in DMSO. Analytical HPLCRt=9.0 min (Flow rate: 0.7 mL/min; Eluent A: water and phosphate buffer50 mmol/dm3, pH 7. Eluent B: acetonitrile; Gradient: 0 min, 2% B; 25min, 50% B; Column: ECONOSPHERE C18, 150 m×4.6 mm).

EXAMPLE 7

Pteroyl amide 11 and pyroglutamic acid 12:

N10-Trifluoroacetylpyrofolic acid 8 (540 mg, 1.04 mmol) was dissolved inaqueous concentrated ammonium hydroxide solution (25 mL). As thereaction proceeded, yellowish solid precipitated. After 14 h, the solidpowder 11 (217 mg, 67%) was isolated by filtration, and washed withwater (20 mL×3), methanol (20 mL×1), and ether (25 mL×2), and driedunder vacuum. ¹H NMR (300 MHz, DMSO-d₆) γ 8.63 (s, 1H, C7-H), 7.61 (d,J=8.3 Hz, 2H, Ar), 6.68 (d, J=8.3 Hz, 2H, Ar), 4.44 (d, J=5.2 Hz, 2H,C9-H2), ¹³C NMR (300 MHz, TFA/inserted DMSO-d₆ tube) γ 172.6, 158.6,151.5, 149.6, 146.7, 145.9, 138.0, 132.6, 130.2, 125.8, 122.9, 53.2.mass spectrum (FAB), 311 (M+); HRMS calcd for C₁₄H₁₃N₇O₂ 312.1209 (MH+),found 312.1205. Decomp. point ˜293° C. Analytical HPLC Rt=9.8 min (Flowrate: 0.7 mL/min; Eluent A: water and phosphate buffer 50 mmol/dm3, pH7; Eluent B: acetonitrile; Gradient: 0 min, 2% B; 25 min, 50% B; Column:ECONOSPHERE C18, 150 mm×4.6 mm). To isolate pyroglutamic acid 12, thesolvent of the above aqueous filtrate was removed by rotary aspirator,and the residue was then purified using preparative HPLC to give 12 (112mg, 84%). ¹H NMR was the same as the one of commercial pyroglutamicacid. [α]D25=−0.74 (c=1.84 in water). MP=154˜158° C. Pyrofolic acid 9(100 mg, 0.236 mmol) in a similar treatment with ammonium hydroxideyielded 11 (11 mg, 15%) and 12 (23 mg, 92%). 11's [α]D25=+0.09 (c=0.46in water).

EXAMPLE 8

Pteroylhydrazide 13:

N10-trifluoroacetylpyrofolic acid 8 (49 g, 94 mmol) was dissolved inDMSO (1,000 mL) with mechanical stirring. To this homogeneous solutionwas added hydrazine (30 mL, 0.94 mol) while maintaining the temperatureat 25° C. During the process, the flask was immersed in a water bath at25° C. and the hydrazine was added slowly in order to moderate a gentleexotherm. The reaction was complete after 8 h as indicated by AnalyticalHPLC, and the mixture was filtered through a pad of celite to remove atrace of solid residue. To the filtrate was then slowly added methanol(1,000 mL) and the resulting precipitated solid was collected byaspirator filtration (or centrifugation), and washed thoroughly withmethanol (200 mL×3) followed by diethyl ether (200 mL×2), to yield crudeproduct 13 (28 g, 91%) after drying 18 h under vacuum. ¹H NMR (300 MHz,D₂O/NaOD) γ 7.91 (s, H, C7-H), 6.98 (d, J=8.5 Hz, 2H, Ar), 6.47 (d,J=8.5 Hz, 2H, Ar), 4.04 (s, 2H, C9-H2). ¹³C NMR (300 MHz, DMSO-d6/HClconc ˜10/1 v/v.) γ 166.3, 158.6, 152.9, 152.8, 152.2, 148.1, 146.9,130.1, 128.6, 117.9, 112.1, 46.0. mass spectrum (FAB), 327 (MH+); HRMScalcd for C₁₄H₁₄N₈O₂ 327.1318, found 327.1307. Decomp. point ˜291° C.Analytical HPLC Rt=14.2 min (Flow rate: 0.7 mL/min; Eluent A: water andphosphate buffer 50 mmol/dm3, pH 7; Eluent B: acetonitrile; Isocratic,5% B; Column: ECONOSPHERE C18, 150 mm×4.6 mm).

EXAMPLE 9

(DL)-methyl folate (γ) 25c (=33a):

To stirred suspension of pyrofolic acid 9 (1.1 g, 2.6 mmol) and methanol(150 mL) was added lithium hexamethyldisilazide (1.0M in THF, 7.8 mL,7.8 mmol) at −10° C. The mixture soon became homogeneous, and thereaction was complete in 9 h at this temperature as indicated byanalytical LC. The basic media was quenched with acetic acid (5 mL) at−78° C. The yellow precipitate was isolated by centrifigation, and thenpurified by preparative reverse phase HPLC to give the methyl folate 25c(804 mg, 70%). 1H NMR (300 MHz, DMSO-d₆) γ 8.60 (s, 1H, C7-H), 7.91 (d,J=7.3 Hz, 1H, N18-H), 7.58 (d, J=8.4 Hz, 2H, Ar), 7.25 (s, 2H, C2-NH2),6.90 (t, J=5.3 Hz, 1H, N10-H), 6.60 (d, J=8.4 Hz, 2H, Ar), 4.45 (d,J=5.3 Hz, 2H, C9-H2), 4.22 (dd, J=7.6, 12.3 Hz, 1H, C19-H), 3.51 (s, 3H,OCH3), 2.47 (t, J=1.7 Hz, 2H, C21-H2), 2.36˜1.88 (m, 2H, C20-H2). ¹³CNMR (300 MHz, DMSO-d₆) γ 174.9, 173.7, 166.7, 162.1, 156.6, 154.8,151.2, 149.0, 148.9, 129.4, 128.3, 122.0, 111.8, 52.9, 51.8, 46.4,30.7,27.3. LRMS (PDMS) (MH+) 455, found 455.5. Decomp. point ˜251° C.Analytical HPLC Rt=10.4 min (Flow rate: 0.7 mL/min; Eluent A: water andphosphate buffer 50 mmol/dm3, pH 7; Eluent B: acetonitrile. Gradient: 0min, 2% B; 25 min, 50% B; Column: ECONOSPHERE C18, 150 mm×4.6 mm).

EXAMPLE 10

Pteroylazide 27 (and N10-Nitroso Pteroylazide 28):

To a stirred suspension of pteroylhydrazide 13 (28 g, 86 mmol) andpotassium thiocyanate (0.41 g, 4.2 mmol) was added ice-coldtrifluoroacetic acid (220 mL). After the solid dissolved, the reactionmixture was cooled to −10° C., followed by slow addition of neattert-butyl nitrite (10 mL, 86 mmol). Monitoring by analytical HPLCindicated that the reaction was complete in 4 h after which time thereaction was warmed to 25° C. In addition to the desired pteroylazide27, Analytical HPLC sometimes showed generation of a small amount (˜10%,due to 90% purity of pteroylazide 27) of N10-nitrosopteroylazide 28,which could be instantly converted into pteroylazide 27 at 25° C. simplyby addition of sodium azide (0.5 equiv. per equiv. of 13 used in thereaction) to the reaction mixture. The solution was then filteredthrough a pad of celite to remove a trace of solid residue. Slowaddition of isopropanol (250 mL) to the stirred filtrate led to anorange powder, which was collected by centrifugation, washed thoroughlywith water (500 mL×3), acetonitrile (500 mL×1), diethyl ether (200mL×2), and finally dried 24 h under vacuum. Multiple cycles of theprecipitation process were used in the trifluoroacetic-acid-isopropanolcombination to obtain material of even higher purity (known impuritiesinclude ˜4% pteroic acid 10, 2% of the aniline apparently resulting fromCurtius rearrangement of pteroylazide 27, ˜2% of pteroylamide 11, and<1% folic acid.). Normally, a purity of more than 90% (determined byHPLC) was achieved after a single purification cycle. The pteroylazide27 (21 g, 72%) thus obtained was stored at −15° C. with protection fromlight. 27: ¹H NMR (300 MHz, DMSO-d₆) γ 8.63 (s, 1H, C7-H), 7.65 (d,J=8.7 Hz, 2H, Ar), 7.57 (m, 1H, N10-H), 6.67 (d, J=8.7 Hz, 2H, Ar), 4.49(d, J=5.8 Hz, 2H, C9-H2). ¹³C NMR (300 MHz, DMSO-d₆) γ 171.0, 158.6,153.9, 152.7, 152.5, 148.3, 146.8, 131.8, 128.3, 117.6, 112.2, 45.8.mass spectrum (FAB), 338 (MH+); HRMS calcd for C₁₄H₁₁N₉O₂ 338.1114,found 338.1110. Decomp. point ˜180° C. Analytical HPLC Rt=18.0 min (Flowrate: 0.7 mL/min; Eluent A: water and phosphate buffer 50 mmol/dm3, pH7; Eluent B: acetonitrile; Gradient: 0 min, 2% B; 25 min, 50% B; Column:ECONOSPHERE C18, 150 mm×4.6 mm). 28 ¹H: NMR (300 MHz, DMSO-d₆) γ 8.74(s, 1H, C7-H), 8.08 (d, J=8.6 Hz, 2H, Ar), 7.91 (d, J=8.6 Hz, 2H, Ar),5.52 (s, 2H, C9-H2). ¹³C NMR (300 MHz, DMSO-d₆) γ 171.6, 160.3, 153.8,153.3, 149.3, 146.7, 145.1, 131.1, 129.0, 128.5, 120.0, 46.9. AnalyticalHPLC Rt=19.2 min (Flow rate: 0.7 mL/min; Eluent A: water and phosphatebuffer 50 mmol/dm3, pH 7; Eluent B: acetonitrile; Gradient: 0 min, 2% B;25 min, 50% B; Column: ECONOSPHERE C18, 150 mm×4.6 mm).

EXAMPLE 11

4-[[(2,4-Diamino-6pteridinyl)methyl]methylamino]benzoyl azide 29:

A solution of 56 (100 mg, 0.31 mmol), diphenyllphosphoryl azide (0.13 mL0.46 mmol), and Et₃N (0.09 mL, 0.62 mmol) in DMSO (5 mL) was stirred for20 h. THF (10 mL) was then added and the precipitate was filtered togive 29 (92 mg, 83%). ¹HNMR is the same as that reported in theliterature.

EXAMPLE 12

Tetramethylguanidinium (L)-methyl folate (γ) 33a (R=TMG-H+):

To a suspension of pteroylazide 27 (5 g, 14.8 mmol) and methyl glutamate(γ) (2.63 g, 16.3 mmol) in DMSO (50 mL) was added neattetramethylguanidine (3.7 mL, 29.7 mmol). The stirred mixture soonbecame homogeneous, and the reaction was complete after 6 h as indicatedby analytical HPLC. The solution was filtered through a pad of celite toremove a trace of solid residue, and acetone (400 ml) was slowly addedto the stirred filtrate. The precipitated solid was then filtered togive the crude tetramethylguanidinium salt of methyl folate (γ) 33a(R=TMG-H+): (6.1 g, 88%) after washing with diethyl ether (50 mL×2), anddrying 18 h under vacuum. ¹H NMR (300 MHz, DMSO-d₆) γ 8.58 (s, 1H,C7-H), 7.55 (d, J=8.4 Hz, 2H, Ar), 6.87 (t, J=5.3 Hz, 1H, N10-H), 6.62(d, J=8.4 Hz, 2H, Ar), 4.41 (d, J=5.3 Hz, 2H, C9-H2), 3.98 (dd, J=7.6,12.3 Hz, 1H, C19-H), 3.49 (s, 3H, OCH3), 2.81 (s, 12H,teramethylguanidine), 2.40˜2.18 (m, 2H, C21-H2), 2.17˜1.78 (m, 2H,C20-H2). mass spectrum (FAB), 454 (M−H+); HRMS calcd for C₂₀H₂₁N₇O₆454.1475, found 454.1445. Spiking experiments in ¹H NMR and analyticalHPLC showed this material to be identical to 25c except for the addedpresence of TMG.

EXAMPLE 13

N-alloc-glutamic acid 47:

To 50 g (340 mmol), of L-glutamic acid in 340 mL of distilled water wasadded 72 g (857 mmol) of sodium bicarbonate in several portions. Afterthe bubbling ceased, 56 mL (510 mmol) of allylchloroformate was addedall at once and the solution left to stir for 18 hours at 25° C. The pHwas adjusted to 1 using concentrated hydrochloric acid and assayed usingpH paper. The solution was transferred to a separatory funnel andextracted 10 times with ethyl acetate. The combined organics were rinsedwith brine and dried over magnesium sulfate. The solvent was removed invacuo to give 57 g (73%) of crude 47 as a colorless syrup.

EXAMPLE 14

3-(3-allyloxycarbonyl-5-oxo-1,3-oxazolan-4-yl)propanoic acid 48:

56.9 g (246 mmol) of 47, 16 g (492 mmol) of paraformaldehyde, 4.7 g(24.6 mmol) of p-toluene sulfonic acid and 1.2L of benzene were combinedin a round-bottom flask equipped with a stir bar and a Dean-Stark trapand heated at reflux for 1 hour. The solvent was removed in vacuo, andthe residue was purified by column chromatography (SiO₂ 4:1 hexane/ethylacetate) to afford 48 (40.6 g, 68%) as a white solid. ¹HNMR (300 MHz,CDC₁₃) γ 5.90 (m, 1H), 5.55 (m, 1H), 5.30 (m, 1H), 4.66 (d, 5.85 Hz,2H), 4.41 (t, 6.01 Hz, 1H), 2.54 (t, 6.95 Hz, 2H), 2.28 (m, 2H). ¹³CNMR(75 MHz, CDC₁₃), γ 177.4, 171.6, 152.9, 131.4, 118.7, 77.6, 66.8, 53.7,28.9, 25.4. mass spectrum (CI), 244 (M+H+, base peak); HRMS calcd forC₁₀H₁₃NO₆ 244.0821, found 244.0823.

EXAMPLE 15

N-alloc, α-allylglutamate 51a:

0.43 g (18.7 mmol) of elemental sodium was added in small pieces to 197mL of allyl alcohol cooled to 0° C. under argon in a flame-dried roundbottom flask. After all the sodium had dissolved, 2.39 g (9.83 mmol) ofoxazolidinone 48 in 20 mL of allyl alcohol was cannulated into thesolution of alkoxide. After stirring for 1 hour at 0° C., the pH wasadjusted to 1 using concentrated hydrochloric acid and pH paper. Thesolution was transferred to a separatory finnel and diluted with andequal volume of water and extracted 6 times with ethyl acetate. Thecombined organics were rinsed with brine and dried over magnesiumsulfate. The solvent was removed in vacuo, and the residue was purifiedby column chromatography (SiO₂, 4:1 hexane/ethyl acetate) to afford 51a(2.1 g, 79%) as a pale yellow oil. ¹HNMR (300 MHz, CDC₁₃) γ 5.85 (m,2H), 5.43 (d, 7.98 Hz, N—H), 5.26 (m, 4H), 4.64 (d, 5.75 Hz, 2H), 4.57(d, 5.33 Hz, 2H), 4.42 (m, 1H), 2.48 (m, 2H), 2.24 (m, 1H), 2.0 (m, 1H).¹³CNMR (75 MHz, CDC₁₃), γ 176.8, 171.4, 155.8, 132.1, 131.0, 118.2,117.2, 65.5, 65.4, 52.8, 29.4, 26.4. mass spectrum (CI), 272 (M+H+, basepeak); HRMS calcd for C₁₂H₁₇NO₆ 272.1134, found 272.1142.

EXAMPLE 16

N-alloc, α-dimethylallylglutamate 51b:

0.54 g (23.5 mmol) of elemental sodium was added in small pieces to 250mL of allyl alcohol cooled to 0° C. under argon in a flame-driedround-bottom flask. After all the sodium had dissolved, 3.0 g (12.3mmol) of 48 in 25 mL of allyl alcohol was cannulated into the solutionof alkoxide. After stirring for 1 hour at 0° C., the pH was adjusted to1 using concentrated hydrochloric acid as monitored using pH paper. Thesolution was transferred to a separatory funnel and diluted with andequal volume of water and extracted 6 times with ethyl acetate. Thecombined organics were rinsed with brine and dried over magnesiumsulfate. The solvent was removed in vacuo, and the remaining liquiddistilled under vacuum to remove the excess dimethylallyl alcohol (0.10mm Hg, b.p. 39° C.). The residue was purified by column chromatography(SiO₂, 4:1 hexane/ethyl acetate) to afford 51b (2.6 g, 71%) as a paleyellow oil. ¹HNMR (300 MHz, CDCl₃) γ 5.90 (m, 1H), 5.42 (d, 8.0 Hz,N—H), 5.30 (m, 3H), 4.64 (d, 7.31 Hz, 2H), 4.57 (d, 5.56, 2H), 4.40 (m,1H), 2.47 (m, 2H), 2.22 (m, 1H), 1.98 (m, 1H), 1.76, (s, 3H), 1.71 (s,3H). ¹³CNMR (75 MHz, CDCl₃), γ 177.0, 171.8, 155.8, 139.5, 132.2, 117.6,117.4, 65.5, 62.1, 52.9, 29.6, 27.0, 25.3, 17.6. mass spectrum (CI), 300(M+H+, base peak); HRMS calcd for C₁₄H₂₁NO₆ 300.1447, found 300.1459.

EXAMPLE 17

2-Aminoethyl folic acid, γ-amide 52 [EDTA-folate (γ)]:

To the crude tetramethylguanidinium (L)-methyl folate (γ) 33a (R=TMG-H+)(2.8 g, 5.95 mmol) was added ethylenediamine (20 mL, 0.3 mol) withstirring at 25° C. The solid gradually dissolved as it reacted with thediamine. The reaction was complete in 3 h as indicated by analyticalHPLC, and filtration gave a clear solution which was then transferred toa well-stirred mixture of acetonitrile and diethyl ether (1:1 v/v, 500mL). The precipitated solid was collected by centrifugation,re-dissolved in water (500 mL), followed by addition of aqueous 5%hydrochloric acid until pH=7.0, which effected precipitation of theproduct. Centrifigation was used to collect the precipitate, which wastriturated with water, and washed thoroughly with water (250 mL×3) toremove any trace of ethylenediamine (¹HNMR was used to assay for thepresence of the diamine.). A yellow solid (2.1 g, 88%) was obtainedafter washing with acetonitrile (100 mL×1), diethyl ether (50 mL×3), anddrying 24 h under vacuum. 52: ¹H NMR (300 MHz, DMSO-d₆/CF₃CO₂D˜10/1 v/v)γ 8.75 (s, 1H, C7-H), 7.66 (d, J=8.6 Hz, 2H, Ar), 6.63 (d, J=8.6 Hz, 2H,Ar), 4.57 (s, 2H, C9-H2), 4.34 (dd, J=4.0, 9.6 Hz, 1H, C19-H), 3.28˜3.13(m, 2H, C24-H2), 2.80 (m, 2H, C25-H2), 2.16(m, 2H, C21-H2), 2.15˜1.85(m, 2H, C20-H2). ¹³C NMR (300 MHz, D₂O/NaOD) γ 178.8, 176.0, 173.1,169.5, 164.0, 155.5, 151.1, 147.3, 147.2, 129.0, 128.2, 121.4, 112.5,55.3, 45.8, 42.0, 39.8, 32.8, 28.0. mass spectrum (FAB), 484 (MH+); HRMScalcd for C₂₁H₂₅N₉O₅ 484.2057, found 484.2062. [α]D25=+4.5,c=0.4 in 1.0NNaOH. Decomp. point ˜278° C. Analytical HPLC Rt=15.4 min (Flow rate: 0.7mL/min. Eluent A: water and phosphate buffer 5 mmol/dm3, pH 7; Eluent B:acetonitrile; Gradient: 0 min, 1% B. 15 min, 10% B. Column: ECONOSPHEREC18, 150 mm×4.6 mm).

EXAMPLE 18

4-[[(2,4-Diamino-6-pteridinyl)methyl]methylamino]benzoic acid 56:

The sodium salt of 34d (equivalent to 200 mg, 0.44 mmol, from theNational Cancer Institute) was dissolved in 5 mL of 0.1M Tris buffer.Carboxypeptidase G ˜5 units) and ZnCl₂ (1 mg) were added and the pH ofthe solution was adjusted to 7.2 with concentrated HCl. The solution wasshaken in a incubator at 38° C. for 2 days, and the mixture (pH 8.4) wasthen adjusted to pH 3.5 with dilute HCl. The precipitated yellow solidwas filtered; washed with H₂O, EtOH, and Et₂O and dried to give theknown acid 56 (152 mg, 96%). ¹HNMR is the same as that reported in C. J.Pouchert Ed., The Aldrich Library of NMR Spectron 2nd ed. AldrichChemical Co. (1983).

EXAMPLE 19

Production of DTPA-Folate:

Dissolve 1.0 g EDA-folate(γ) (2.1 mmol) in 50 ml DMSO by bath sonicationovernight. Then the dark yellow solution was slowly added to a stirringsuspension of 20 g DTPA dianhydride (5.6 mmol) in 10 mL anhydrous DMSO.The mixture became homogeneous by the end of addition. Analytical HPLCshowed the absence of the starting material EDA-folate after 30 min. atwhich time the reaction mixture was filtered through a pad of celite toremove traces of solid residue. After reducing the temperature of thereaction mixture with ice bath, 10 mL 2.4 N NaOH was added to quench thereaction and to neutralize the solution. The resulting precipitatecontaining the majority of the DTPA-folate(γ) produced was separated bycentrifugation from the supernatant. The yellow pellet was then washedwith 100 mL acetonitrile, dissolved in 50 mL water, and the pH of thesolution was adjusted to 7 with concentrated HCL. After filtration toremove the solid residue, the clear orange solution was directlypurified on a LiChroprep C-189 reversed phase MPLC column (310 mm×25 mm,45-60 μm) using 10 mM ammonium bicarbonate buffer as the eluant(t_(r)=20-35 min, flow rate: 1 mL/min). The collected product wasconcentrated by vacuum and further purified by preparative HPLC on theMicrosorb C-18 column with a gradient (eluant A: 10 mM ammoniumbicarbonate buffer, pH 7.4; eluant B: acetonitrile; gradient: 0 min, 4$B; 10 min, 12% B; 15 min, 15% B; flow rate: 10 mL/min; t_(r)−6.3 min) toremove residual bis-conjugated side product and to achieve 0.85 gDTPA-folate after lyophilization with a purity above 99% and a yield of47%. Analytical HPLC on the Econosphere C-18 reversed phase column (150mm×4.6 mm) revealed a single peak with a retention time of 11.74 min(eluant: 10 mM tetrabutylammonium phosphate buffer, pH 7, 75%,acetonitrile, 25%; flow rate: 0.7 mL/min). ¹H NMR (300 MHz, D₂O) γ 8.46(s, 1H, C7-H), 7.41 (d, J=8.3 Hz, 2H, Ar), 6.34 (d, J=8.3 Hz, 2H, Ar),4.24 (dd, J=4.4, 8.4 Hz, 1H, C19-H), 4.15 (s, 2H, C9-H₂), 3.53 (s, 4H),3.47 (s, 2H), 3.42 (s, 2H), 3.18˜2.97 (overlap, 14H), 2.28 (m, 2H,C22-H₂), 2.28˜1.83 (m, 2H, C21-H₂). ¹³CNMR (75 MHz, D₂O) γ 178.9, 178.0,175.9, 175.7, 173.6, 173.2, 169.0, 164.8 (C4), 154.4 (C2), 152.8 (C8a),150.2 (C11), 148.3, 148.2, 128.9 (C13/15), 126.5 (C4a), 120.7 (C14),111.7 (C12/16), 58.6, 58.2, 57.9, 55.6, 55.4, 51.9, 51.6, 51.1, 50.6,45.4 (C9), 38.8 (C25), 38.5 (C26), 32.8 (C22), 28.1 (C21).High-resolution MS (Matrix Assisted Laser Desorption Ionization)C₃₅H₄₆N₁₂O₁₄ (MH⁺) 857.830, found 587.853.

EXAMPLE 20

Radiotracer Synthesis:

The ¹¹¹In-DTPA-folate radiopharmaceutical was obtained in highradiochemical yield by ligand exchange from ¹¹¹In-citrate. Briefly,¹¹¹In³⁺ (0.2-5.4 mCi) in Hcl (0.05 M; 2.55 μL) was transferred to a testtube and buffered by addition of 200 μL 3% aqueous sodium citrate. Theresulting ¹¹¹In-citrate was mixed with 300-350 μg DTPA-folate in water(2 mg/mL; pH 7-8). Two to 24 hours later, the radiochemical purity of hte ¹¹¹In-DTPA-folate was determined by TLC on C-18 reversed phase plateseluted with methanol and consistently found to exceed 98%(¹¹¹InDTPA-folate R_(f)=0.8; ¹¹¹In-citrate R_(f)=0.0). The¹¹¹In-DTPA-folate product was diluted with normal saline, as needed,prior to use in the animal biodistribution experiments.

EXAMPLE 21

Cellular Uptake of ¹¹¹In-DTPA-Folate:

Cultured KB and A549 cells in 35 mm dishes were incubated with 100 nM¹¹¹In-citrate or ¹¹¹In-labeled DTPA-folate in 1 mL folate-deficientmedium at room temperature for various lengths of time. The cells werethen washed with 3×1 mL PBS and suspended in 1 mL PBS by scraping. Theamount of cell-associated radioactivity was determined using anautomatic gamma-counter. Cellular protein content was measured by thebicinchoninic acid assay. In folate competition experiments, the sameprotocol was used except that 1 mM folic acid was included in theincubation medium.

EXAMPLE 22

Biodistribution and Imaging Studies with ¹¹¹In-DTPA-Folate:

All animal studies were carried out in accordance with proceduresapproved by the Purdue Animal Care and Use Committee. Thebiodistribution of ¹¹¹In-DTPA-folate and ⁶⁷Ga-DF-folate were determinedI normal male Sprague-Dawley rats following intravenous injection underdiethyl ether anesthesia, as described previously (Tsang et al., 1994).In addition, a gamma scintillation image of a female athymic mouse Nu/Nu(22 g) was obtained using a PhoGamma 37GP camera fitted with a 300 keVparallel hold collimator. The animal used for this imaging study wasmaintained for three weeks on folate-free diet (to reduce its serumfolate to a level near that of human serum) and had been implantedsubcutaneously in the interscapular region with human KB tumor cells.Tumor mass at the time of the imaging study was approximately 0.25 g.The animal was imaged 1 hour following intravenous administration of¹¹¹In-DTPA-folate (200 μCi in 0.1 mL) via the femoral vein under diethylether anesthesia. To promote clearance of radiotracer from the urinarybladder, 1.5 mL sterile saline was administered by i.p. injectionimmediately following he intravenous radiotracer injection. For imaging,the mouse was re-anesthetized with ketamine (60 mg/kg) and xylazine (6mg/kg) immediately prior to the image acquisition period. Totaladministered mass of DTPA-folate conjugate was 13 μg (0.57 mg/kg).

Affinities of FITC-EDA-Folate for Cell Surface Receptors:

To determine the affinities of EDA-folate for cell surface folatereceptors, the α- and γ-isomers were labeled with the fluorophore FITCand incubated with KB cells overexpressing the receptor. As shown inFIG. 3, 1.6 nM FITC-EDA-folate(γ) was required to reach 50% maximalbinding, similar to that of folic acid and DF-folate(γ). Excess folicacid in the incubation medium effectively competed with the receptorbinding of FITC-EDA-folate(γ). On the other hand, the α-isomer ofFITC-EDA-folate had virtually no affinity for the cell surfacereceptors. The low level of non-specific uptake observed with bothisomers was probably due to the hydrophobicity of the FITC conjugates.

What is claimed is:
 1. A compound of the formula

wherein Q is OH or NH₂, Y is hydrogen, nitroso, C₁-C₄ alkyl, C₁-C₄alkanoyl, C₁-C₄ halosubstituted alkanoyl and Z is NHNH₂, or a group offormula

provided that when Z is

Y is other than acetyl or trifluroacetyl.
 2. A method of preparing acompound of the formula

wherein Q is OH or NH₂, Y is hydrogen, nitroso, C₁-C₄ alkyl, C₁-C₄alkanoyl, or C₁-C₄ halosubstituted alkanoyl, said method comprising thestep of reacting a compound of formula

wherein X is H or trifluroacetyl and Y′ is hydrogen, C₁-C₄ alkyl, C₁-C₄alkanoyl or C₁-C₄ halosubstituted alkanoyl, with hydrazine.